Curcumin facilitates a transitory cellular stress response in Trembler-J mice.

We have previously shown that oral administration of curcumin significantly decreases the percentage of apoptotic Schwann cells and partially mitigates the severe neuropathy phenotype of the Trembler-J (Tr-J) mouse model in a dose-dependent manner. Here we compared the gene expression in sciatic nerves of 2-week-old pups and adult Tr-J with the same age groups of wild-type mice and found a significant increase in gene expression for hypoxia, inflammatory response and heat-shock proteins, the latter specifically the Hsp70 family, in Tr-J mice. We also detected an activation of different branches of unfolded protein responses (UPRs) in Tr-J mice. Administering curcumin results in lower expression of UPR markers suggesting it relieves endoplasmic reticulum (ER) cell stress sensors in sciatic nerves of Tr-J mice while the level of heat-shock proteins stays comparable to untreated Tr-J mice. We further tested if Hsp70 levels could influence the severity of the Tr-J neuropathy. Notably, reduced dosage of the Hsp70 strongly potentiates the severity of the Tr-J neuropathy, though the absence of Hsp70 had little effect in wild-type mice. In aggregate, these data provide further insights into the pathological disease mechanisms caused by myelin gene mutations and further support the exploration of curcumin as a therapeutic approach for selected forms of inherited neuropathy and potentially for other genetic diseases due to ER-retained mutants.

The Texas Immuno-Oncology Biorepository, a statewide biospecimen collection and clinical informatics system to enable longitudinal tumor and immune profiling.

A detailed understanding of the molecular and immunological changes that occur longitudinally across tumors exposed to immune checkpoint inhibitors is a significant knowledge gap in oncology. To address this unmet need, we created a statewide biospecimen collection and clinical informatics system to enable longitudinal tumor and immune profiling and to enhance translational research. The Texas Immuno-Oncology Biorepository (TIOB) consents patients to collect, process, store, and analyze serial biospecimens of tissue, blood, urine, and stool from a diverse population of over 100,000 cancer patients treated each year across the Baylor Scott & White Health system. Here we sought to demonstrate that these samples were fit for purpose with regard to downstream multi-omic assays. Plasma, urine, peripheral blood mononuclear cells, and stool samples from 11 enrolled patients were collected from various cancer types. RNA isolated from extracellular vesicles derived from plasma and urine was sufficient for transcriptomics. Peripheral blood mononuclear cells demonstrated excellent yield and viability. Ten of 11 stool samples produced RNA quality to enable microbiome characterization. Sample acquisition and processing methods are known to impact sample quality and performance. We demonstrate that consistent acquisition methodology, sample preparation, and sample storage employed by the TIOB can produce high-quality specimens, suited for employment in a wide array of multi-omic platforms, enabling comprehensive immune and molecular profiling.

Expanded CTG repeats within the DMPK 3' UTR causes severe skeletal muscle wasting in an inducible mouse model for myotonic dystrophy.

Severe skeletal muscle wasting is the most debilitating symptom experienced by individuals with myotonic dystrophy type 1 (DM1). We present a DM1 mouse model with inducible and skeletal muscle-specific expression of large tracts of CTG repeats in the context of DMPK exon 15. These mice recapitulate many findings associated with DM1 skeletal muscle, such as CUG RNA foci with Muscleblind-like 1 (MBNL1) protein colocalization, misregulation of developmentally regulated alternative splicing events, myotonia, characteristic histological abnormalities, and increased CUGBP1 protein levels. Importantly, this DM1 mouse model recapitulates severe muscle wasting, which has not been reported in models in which depletion of MBNL1 is the main feature. Using these mice, we discovered previously undescribed alternative splicing events that are responsive to CUGBP1 and not MBNL, and these events were found to be misregulated in individuals with DM1. Our results indicate that increased CUGBP1 protein levels are associated with DMPK-CUG RNA expression, suggesting a role for CUGBP1-specific splicing or cytoplasmic functions in muscle wasting.

Upregulation of hypoxia-inducible factor (HIF)-1alpha and HIF-2alpha in leptomeningeal vascular malformations of Sturge-Weber syndrome.

Cutaneous and leptomeningeal vascular malformations are hallmarks of the Sturge-Weber Syndrome (SWS), resulting in chronic ischemic tissue damage. The mechanisms underlying the pathobiology of these progressive lesions are unknown. Aberrant expression of angiogenic factors has been implicated in the genesis and maintenance of vascular malformations. To assess the role of angiogenesis in SWS vascular lesions we determined the expression of key angiogenic factors by immunohistochemistry and in situ hybridization in 8 SWS patients (age: 8 months to 18 years). We observed increased expression of vascular endothelial growth factor (VEGF), its cognate receptors VEGFR-1, VEGFR-2, and neuropilin (NP)-1 as well as Tie2 in leptomeningeal SWS blood vessels. Intriguingly, these factors are known to be transcriptionally induced by hypoxia-inducible factor (HIF). The HIF system has emerged as the key regulatory system of responses to hypoxia. Immunohistochemical analysis demonstrated markedly elevated nuclear HIF-1alpha and HIF-2alpha protein levels in SWS vessels. Concomitantly, SWS vessels revealed signs of enhanced endothelial cell (EC) turnover as evidenced by increased EC proliferation and apoptosis. Thus, in terms of angiogenesis, vascular malformations in SWS are not static lesions but constitute dynamic structures. Our observation of a dysregulated HIF-alpha expression in SWS vessels are in agreement with recent findings that EC-specific HIF activation provides a setting which supports and sustains angiogenesis and could be of potential use for developing therapeutic strategies to treat these currently incurable lesions.

Suppression of Rac activity induces apoptosis of human glioma cells but not normal human astrocytes.

Tumors of glial origin such as glioblastoma multiforme (GBM) comprise the majority of human brain tumors. Patients with GBM have a very poor survival rate, with an average life expectancy of <1 year. We asked whether we could identify a survival pathway in high-grade glioma and oligodendroglioma cells that when suppressed, would induce apoptosis of these tumor cells but not of normal human adult astrocytes. To identify these pathways, we selectively suppressed the activity of a number of proteins (Ras, Rac1, Akt1, RhoA, c-jun, and MEK1/2) hypothesized to play roles in cell survival. We found that suppression of Rac1, a small GTP-binding protein, inhibited survival and produced apoptosis in three human glioma cell lines (U87, U343, and U373). Serum induced the activity of Rac1 and the activity or phosphorylation state of p21-activated kinase 1 and c-Jun NH(2)-terminal kinase (JNK), two intracellular targets of Rac1. Suppression of Rac1 also induced apoptosis in 19 of 21 short-term cultures of human primary cells from grades II and III oligodendroglioma and grade IV glioblastoma that varied in p53, epidermal growth factor receptor, epidermal growth factor receptor vIII, MDM2, and p16/p19 mutational or amplification status. In contrast, inhibition of Rac1 activity did not induce apoptosis of normal primary human adult astrocytes. In both established glioma cell lines and primary glioma cells, apoptosis induced by the inhibition of Rac was partially rescued by activated mitogen-activated protein kinase kinase 1, an activator of JNK, suggesting that JNK functions downstream of Rac1 in glioma cells. These results indicate that Rac1 regulates a major survival pathway in most glioma cells, and that suppression of Rac1 activity stimulates the death of virtually all glioma cells, regardless of their mutational status. Agents that suppress Rac1 activity may therefore be useful therapeutic treatments for malignant gliomas.

Phenotypic differences between peripheral myelin protein-22 (PMP22) and myelin protein zero (P0) mutations associated with Charcot-Marie-Tooth-related diseases.

Mutations in the genes for peripheral myelin protein-22 (PMP22) and myelin protein zero (P0) cause human hereditary neuropathies with varying clinical and pathological phenotypes. In this study, we examine the effects of representative disease-causing mutations on the subcellular distribution of their corresponding PMP22- and P0-enhanced green fluorescent protein (EGFP) fusion proteins. In transiently transfected HeLa and 293 cells, we find that wild-type P0-EGFP and PMP22-EGFP are efficiently synthesized and transported through the secretory pathway to the plasma membrane. The P0-EGFP and PMP22-EGFP mutants can be classified into several groups: those that are transported to the plasma membrane as in the majority of P0 mutants; those that are retained in the endoplasmic reticulum as in the majority of PMP22 mutants; and those that are a mixture of the two. In addition, several of these disease-causing mutations are associated with the development of abnormal intracellular cytoplasmic structures that we have previously identified as either intracellular myelin figures or aggresomes. Our studies indicate that different types of PMP22 and P0 mutations are associated with specific intracellular chaperone proteins, including calnexin and BiP, and that these associations can be altered by glycosylation. These findings indicate that the various P0 and PMP22 mutants may exert their pathogenic effects in different subcellular compartments and by different mechanisms in the mammalian cell.

Continuous electrocorticogram epileptiform discharges due to brain gliosis.

Cortical dysplasia is known to produce continuous epileptiform discharges (CEDs) on electrocorticogram (EcoG) and EEG recordings. The authors studied the incidence of CEDs on ECoGs and correlated this data with pathologic findings. Thirty ECoGs were reviewed that were performed on patients with parietal or occipital lobe epilepsy operated on since 1960. CED was classified as: (1) continuous or semicontinuous rhythmic spikes or sharp waves at frequencies ranging from 2 to 8 Hz, and (2) repetitive bursts of rhythmic polyspike activity lasting 2 to 10 s. All nontumoral pathologic specimens were reviewed. Epileptiform activity was classified using the following criteria: focal (one gyrus), regional (two gyri), lobar (three gyri), bilobar, or multilobar. Pathologic examination showed gliosis in eight specimens, focal cortical dysplasia in five specimens, tumoral lesions in eight specimens, and other pathology in nine specimens. CED was found in 11 ECoGs. In seven pathology specimens, significant gliosis was shown, and in the remaining four specimens, a dysplastic lesion was diagnosed. Epileptiform activity was widespread (lobar, bilobar, or multilobar) when gliosis or focal cortical dysplasia was present. Absence of epileptiform activity or a focal/regional distribution was found in tumors and other lesions. These data suggest that extensive gliotic lesions are highly epileptogenic and produce CEDs, which are morphologically undistinguishable from those produced by focal cortical dysplasia.

Molecular mechanisms, diagnosis, and rational approaches to management of and therapy for Charcot-Marie-Tooth disease and related peripheral neuropathies.

During the last decade, 18 genes and 11 additional loci harboring candidate genes have been associated with Charcot-Marie-Tooth disease (CMT) and related peripheral neuropathies. Ten of these 18 genes have been identified in the last 2 years. This phenomenal pace of CMT gene discovery has fomented an unprecedented explosion of information regarding peripheral nerve biology and its pathologic manifestations in CMT. This review integrates molecular genetics with the clinical phenotypes and provides a flowchart for molecular-based diagnostics. In addition, we discuss rational approaches to molecular therapeutics, including novel biologic molecules (eg, small interfering ribonucleic acid [siRNA], antisense RNA, and ribozymes) that potentially could be used as drugs in the future. These may be applicable in attempts to normalize gene expression in cases of CMT type 1A, wherein a 1.5 Mb genomic duplication causes an increase in gene dosage that is associated with the majority of CMT cases. Aggresome formation by the PMP22 gene product, the disease-associated gene in the duplication cases, could thus be avoided. We also discuss alternative therapeutics, in light of other neurodegenerative disorders, to disrupt such aggresomes. Finally, we review rational therapeutic approaches, including the use of antioxidants such as vitamin E, coenzyme Q10, or lipoic acid to relax potential oxidative stress in peripheral nerves, for CMT management.

Reprogramming tumor-infiltrating dendritic cells for CD103+ CD8+ mucosal T-cell differentiation and breast cancer rejection.

Our studies showed that tumor-infiltrating dendritic cells (DC) in breast cancer drive inflammatory Th2 (iTh2) cells and protumor inflammation. Here, we show that intratumoral delivery of the ß-glucan curdlan, a ligand of dectin-1, blocks the generation of iTh2 cells and prevents breast cancer progression in vivo. Curdlan reprograms tumor-infiltrating DCs via the ligation of dectin-1, enabling the DCs to become resistant to cancer-derived thymic stromal lymphopoietin (TSLP), to produce IL-12p70, and to favor the generation of Th1 cells. DCs activated via dectin-1, but not those activated with TLR-7/8 ligand or poly I:C, induce CD8+ T cells to express CD103 (αE integrin), a ligand for cancer cells, E-cadherin. Generation of these mucosal CD8+ T cells is regulated by DC-derived integrin αvß8 and TGF-ß activation in a dectin-1-dependent fashion. These CD103+ CD8+ mucosal T cells accumulate in the tumors, thereby increasing cancer necrosis and inhibiting cancer progression in vivo in a humanized mouse model of breast cancer. Importantly, CD103+ CD8+ mucosal T cells elicited by reprogrammed DCs can reject established cancer. Thus, reprogramming tumor-infiltrating DCs represents a new strategy for cancer rejection.

Oral curcumin mitigates the clinical and neuropathologic phenotype of the Trembler-J mouse: a potential therapy for inherited neuropathy.

Mutations in myelin genes cause inherited peripheral neuropathies that range in severity from adult-onset Charcot-Marie-Tooth disease type 1 to childhood-onset Dejerine-Sottas neuropathy and congenital hypomyelinating neuropathy. Many myelin gene mutants that cause severe disease, such as those in the myelin protein zero gene (MPZ) and the peripheral myelin protein 22 gene (PMP22), appear to make aberrant proteins that accumulate primarily within the endoplasmic reticulum (ER), resulting in Schwann cell death by apoptosis and, subsequently, peripheral neuropathy. We previously showed that curcumin supplementation could abrogate ER retention and aggregation-induced apoptosis associated with neuropathy-causing MPZ mutants. We now show reduced apoptosis after curcumin treatment of cells in tissue culture that express PMP22 mutants. Furthermore, we demonstrate that oral administration of curcumin partially mitigates the severe neuropathy phenotype of the Trembler-J mouse model in a dose-dependent manner. Administration of curcumin significantly decreases the percentage of apoptotic Schwann cells and results in increased number and size of myelinated axons in sciatic nerves, leading to improved motor performance. Our findings indicate that curcumin treatment is sufficient to relieve the toxic effect of mutant aggregation-induced apoptosis and improves the neuropathologic phenotype in an animal model of human neuropathy, suggesting a potential therapeutic role in selected forms of inherited peripheral neuropathies.

Curcumin treatment abrogates endoplasmic reticulum retention and aggregation-induced apoptosis associated with neuropathy-causing myelin protein zero-truncating mutants.

Mutations in MPZ, the gene encoding myelin protein zero (MPZ), the major protein constituent of peripheral myelin, can cause the adult-onset, inherited neuropathy Charcot-Marie-Tooth disease, as well as the more severe, childhood-onset Dejerine-Sottas neuropathy and congenital hypomyelinating neuropathy. Most MPZ-truncating mutations associated with severe forms of peripheral neuropathy result in premature termination codons within the terminal or penultimate exons that are not subject to nonsense-mediated decay and are stably translated into mutant proteins with potential dominant-negative activity. However, some truncating mutations at the 3' end of MPZ escape the nonsense-mediated decay pathway and cause a mild peripheral neuropathy phenotype. We examined the functional properties of MPZ-truncating proteins that escaped nonsense-mediated decay, and we found that frameshift mutations associated with severe disease cause an intracellular accumulation of mutant proteins, primarily within the endoplasmic reticulum (ER), which induces apoptosis. Curcumin, a chemical compound derived from the curry spice tumeric, releases the ER-retained MPZ mutants into the cytoplasm accompanied by a lower number of apoptotic cells. Our findings suggest that curcumin treatment is sufficient to relieve the toxic effect of mutant aggregation-induced apoptosis and may potentially have a therapeutic role in treating selected forms of inherited peripheral neuropathies.

An 8.5-kb segment of the PMP22 promoter responds to loss of axon signals during Wallerian degeneration, but does not respond to specific axonal signals during nerve regeneration.

Altered expression of the PMP22 gene causes Charcot-Marie-Tooth disease type 1A (CMT1A) and hereditary neuropathy with liability to pressure palsies (HNPP). We have examined the promoter activity of 8.5 kb upstream of the first coding exon of the rat peripheral myelin protein-22 (rPmp22) gene in transgenic mice. We found that the -8.5 kb rPmp22/chloramphenicol acetyl transferase (CAT)/beta-galactosidase (lacZ) construct directs reporter gene expression in a weakly developmental and tissue-specific pattern, consistent with the expression pattern of the endogenous Pmp22 gene. The -8.5 kb rPmp22/CAT/lacZ transgene responds to loss of axonal signals during Wallerian degeneration but unlike the endogenous Pmp22 gene, the transgene fails to respond to axonal signals during nerve regeneration after a sciatic nerve crush injury. In conclusion, the function of the -8.5 kb rPmp22/CAT/lacZ transgene suggests that there are separable regulatory elements in the rPmp22 gene that respond differently to axonal signals received by Schwann cells during nerve development, and during remyelination.

The Anatomy and Cell Biology of Peripheral Myelin Protein-22.

The gain of function phenotypes exhibited by the heterozygous Tr, Tr-J, and CMT1A mutations indicate that these mutations interfere with more than the function of a single PMP22 allele. The identification of proteins that interact with PMP22 and that are sensitive both to stoichiometry and the effects of the mutations could provide important leads to a unified hypothesis to explain the riddle of the PMP22-related neuropathies.